All living cells are capable of extracting information from their micro-environment and mounting appropriate responses to a variety of associated challenges. The underlying signal transduction networks can be quite complex, necessitating for their unraveling a combination of sophisticated computational modeling and precise experimentation. Unfortunately, current computational and experimental analysis of cell signaling frequently suffers from such pitfalls as isolation of a pathway from surrounding signaling network, disregard of the cell-cell variability in the signaling outputs or studying signaling out of the context provided of by cell-cell communication in the native tissues. This renewal proposal is aimed at providing a framework for addressing these research limitations through development of novel methods and tools, and putting forward a detailed plan of a more realistic integrative analysis of signaling in response to a chemokine, tumor necrosis factor (TNF). A particular emphasis of our analysis will be on understanding of the information transfer properties of signaling pathways and its role in defining the precision of the phenotypic outcomes, including regulation of gene expression. The results of the analysis will provide a new platform for investigation of the relationship between the single cell and population responses and drive the development of the information theory based understanding of intracellular signal processing and cell communication. We anticipate that the quantitative understanding of the complexity of signaling cross-talk, regulation of diversity of cell responses to the same stimulus and nuances of cell-cell communication will facilitate development of a more realistic framework for understanding of the human disease, including functioning of the immune system, and drug development aimed at regulation of the NF-kappaB and JNK signaling. PUBLIC HEALTH RELEVANCE: Cells respond to their external environment by extracting information out of chemical signals they receive from other cells. This leads to changes in protein activity and gene expression, ultimately deciding the function and fate of the cell. These responses are complicated by differences between individual cells as well as communication between cells. Understanding the responses may benefit from analyses that combine experiment and mathematical modeling. In this context, we propose to study in detail two signals induced by inflammation in mammalian cells. Through such analyses we anticipate to uncover novel principles and mechanisms by which cells respond to external stimuli.